BIOLOGY OF REPRODUCTION 50, 225-232 (1994)

Ovarian Follicular Growth and Development in Mammals'

J.E. FORTUNE 2

Department and Section of Physiology, Cornell University, Ithaca, New York 14853

ABSTRACT
Evidence from several species indicates that the initial stages of follicular growth proceed very slowly. In contrast, the stages
after antrum formation are much more rapid. Atresia seems to be most prevalent as follicles approach the size at which they
could be recruited for potential ovulation. Although most follicles become atretic around that stage, a few are recruited into a
cohort or wave of follicles that continue to grow beyond the stage at which atresia normally occurs. Next, a species-specific
number of follicles is selected for dominance. In some species (e.g. rats, primates, pigs), dominant follicles develop only during
the follicular phase and are thus destined for ovulation. In another group of species (e.g. cattle, sheep, horses), recruitment,
selection, and dominance occur at regular intervals, but only the dominant follicle present during the follicular phase ovulates.
There is evidence that the mechanism that allows some follicles to be recruited for potential dominance/ovulation is a small
elevation in basal FSH that, by chance, occurs around the time the follicle would normally begin atresia. Some recruited follicles
are saved from atresia for only a short time. Only the dominant follicle(s) selected from among the recruited follicles grows to
ovulatory or near-ovulatory size. What determines which follicle(s) becomes dominant is not known, but dominance appears to
be maintained by negative feedback effects of products of the dominant follicle on circulating FSH.
Selection and dominance are accompanied by progressive increases in the ability of thecal cells to produce androgen and
granulosa cells to aromatize androgen to estradiol. A small rise in plasma LH during the follicular phase seems necessary for these
changes. Results of experiments with rats and cattle indicate that the enhanced estrogen-synthesizing capacity of ovulatory fol-
licles is mediated by increases in messenger RNA for appropriate steroidogenic enzymes. The LH/FSH surge radically changes
follicular steroidogenesis by decreasing androgen and estradiol production, and increasing follicular capacity to secrete proges-
terone. These shifts in steroidogenic patterns are not completely consistent with reported changes in mRNA levels for steroido-
genic enzymes.
Although much is now known about the dynamics of follicular development and about functional changes as follicles differ-
entiate, some central questions about follicular development remain to be answered. We still do not know why follicles leave
the resting pool and why only some recruited follicles are selected for dominance. In addition, there is still much to be learned
about the roles of interactions between the gonadotropins and follicular cells, and between follicular cells and the oocyte, in
the various stages of follicular differentiation.

INTRODUCTION common mechanisms that regulate these processes across

Before reproductive senescence, mammalian ovaries have
a pool of primordial follicles, each consisting of an oocyte
arrested in prophase I of meiosis and a single layer of flat-
tened granulosa cells. This pool develops during fetal life
in some species (e.g. primates, ruminants), but in others it
develops during the early neonatal period (e.g. rodents,
rabbits) [1, 2]. Once the cohort of primordial follicles has
been established, follicles gradually and continually leave
the resting pool to begin growth. The nature of the signals
that initiate growth, and the mechanisms that ensure that
follicles leave the resting pool gradually, are unknown. Once
a follicle begins to grow, growth seems to be continuous
until the follicle meets one of two fates-ovulation or atre-
sia. It is well known that very few follicles that begin growth
successfully ovulate; most die before reaching that stage.
The purpose of this paper is to review the mechanisms
that regulate the later stages of follicular development, i.e.,
the growth of large antral follicles and the selection and
differentiation of a species-specific number of follicles for
ovulation during each reproductive cycle, and to identify
'Research from the author's laboratory presented in this manuscript was sup-
ported by grants from the USDA (90-37240-5715) and the NIH (HD14584).
2Correspondence. FAX: (607) 253-3851.
species. However, these later stages of follicular develop-
ment are best understood in the context of the patterns of
follicular growth that precede them. Therefore, I will first
briefly consider patterns of follicular growth and atresia over
the whole continuum of follicular development; then focus
on the phenomena of follicular recruitment, selection, and
dominance; and finally consider the endocrinological dif-
ferentiation of dominant follicles.
TEMPORAL PATTERNS OF FOLLICULAR GROWTH AND
ATRESIA
Although it has been commonly believed that atresia can
occur at any stage of follicular development, and this is
probably true, careful analysis of patterns of follicular growth
and atresia in a number of species have revealed that atre-
sia is not equally prevalent across all stages of follicular
development. Hirshfield [1] has analyzed patterns of follic-
ular growth and atresia in a particularly intriguing fashion
by considering follicular development as a series of gran-
ulosa cell generations, with a generation defined as the length
of time required for the number of granulosa cells visible
in the largest cross section through the follicle to double.
When the growth of rat follicles is analyzed in this way,
some interesting patterns emerge. First, the initial granu-

225
226 FORTUNE

4 TABLE 1. Characteristics of bovine ovarian follicles during

development.*
ATRESIA
Granulosa cell Follicular Estimated # Percent Developmental
4, generation diameter of granulosa atretic time
(approximate) (mm) cells/section follicles (days)
1 2 3 4 5 78910
6 CL
Ii hal 3I in M d 4i 6 0.13-0.28 168 2 15
more than 30 days Ed 5d 4d 4d2.5d2d 8 0.29-0.67 736 7 12
10 0.68-1.52 2,708 39 3
11 1.53-3.67 5,558 30 4
*GC MATURATION 12 3.68-8.57 11,585 67 8
13 >8.57 22,387 60 -
FIG. 1. Schematic diagram of the temporal progression of follicular
growth in rats, showing that the initial stages of follicular development oc- *Data adapted from Lussier et al. [3].
cur much more slowly than the final stages, and that most follicular atresia
occurs during a few granulosa cell generations. The numbers and filled
circles above the line indicate granulosa cell generations (time required for
doubling of granulosa cells in the largest cross section though the follicle). in follicles that are 2-10 mm in diameter. In humans, the
GC, granulosa cell. (Figure provided by Dr. A. N. Hirshfield). initial stages of follicular development take several months,
whereas development from 2-10 mm is estimated to take
only about 20 days [6].
losa cell generations (i.e., the first stages of follicular growth) Therefore, for these three species-rats, cattle, and hu-
proceed much more slowly than the final stages of follic- mans-the incidence of atresia is not evenly distributed
ular development (Fig. 1). Second, although little atresia across follicular development, but rather most atresia oc-
occurs during the first 7 granulosa cell generations, most curs during a short temporal period of follicular develop-
follicles become atretic during the 8th or 9th generation. ment and during specific granulosa cell generations that
This suggests that follicular growth and development up to immediately precede the penultimate generation. What these_
a certain point can occur readily in the presence of normal observations imply is that follicular development can pro-
basal concentrations of gonadotropins, metabolic hor- ceed rather easily, without much follicular loss, under basal
mones, and growth factors, but that when follicles reach the
8th-9th granulosa cell generation they reach the end of their
normal life span under those basal conditions. Theoreti-
cally, then, only follicles that are exposed to specific, ad-
ditional signals will continue to the 10th granulosa cell gen-
eration and ovulate. Hirshfield has expressed this concept
as follicles "hitting a brick wall" and being unable to sur- L
mount it unless they receive particular signals at precisely
the time when they reach this normal stopping point in
their development. The data and reasoning that support these E
ideas are presented in more detail in a recent review by I)CU
Hirshfield [1]. :3
S_
0)
Does this concept of granulosa cell generations and of
..
a natural barrier to the continuation of follicular develop- Primates
ment apply to species other than rats? Lussier et al. [3] have Pigs
carefully analyzed patterns of follicular growth and atresia Rats
in bovine ovaries. If we assume, on the basis of the histo-
logical data of Rajakoski [4], that bovine primordial follicles
have about 5 granulosa cells in their largest-diameter cross
section, and apply the concept of granulosa cell generations
to the data of Lussier et al. [3], then it becomes evident that
I_
0

in cattle atresia is heaviest just before the final stages of

follicular development (granulosa cell generations) (Table
1). The final stages of bovine follicular development occur
comparatively rapidly [3, 5], whereas the rate of growth of
follicles < 0.5 mm in diameter is estimated to be much
slower [5]. Therefore, in cattle, as in rats, the vast majority
of follicular atresia occurs over the course of only a few
granulosa cell generations near the end of follicular de-
velopment (generations 10-13). Likewise, for human fol-
licles, Gougeon [6] has reported the highest rates of atresia
,2777
Luteal Phase
a
Follicular Phase
FIG. 2. Schematic diagrams of two patterns of follicular development
during mammalian reproductive cycles. The lines represent changes in fol-
licular diameter as follicles grow or regress; the asterisks denote ovulation.
The upper panel depicts a pattern exemplified by cattle, horses, and sheep,
in which dominant, ovulatory-size follicles develop throughout the cycle.
The lower panel depicts a pattern observed in species such as rats, pri-
mates, and pigs, in which dominant, ovulatory-size follicles develop only
during the follicular phase.
 FOLLICULAR DEVELOPMENT IN MAMMALS 227

TABLE 2. Characteristics of follicles collected from heifers during the first wave of follicular development.

Day of estrous Follicular Follicular Follicular fluid

cycle No. follicles diameter fluid estradiol androstenedione
(N = 4 heifers/day) >6 mm per heifer (mm) (ng/ml) (ng/ml)
Day 3 8.0 ± 1.28 7.4 0.21 58.5 13.58 96.4 ± 17.30
(N = 32 follicles)

Day 7 3.0 04 b 10.3 ± 1.3 79.8 ±+35.0' 9.7 2.4b

(N = 12 follicles)

Dominant 1.0 0.0 16.1 ± 1.0 201.2 ± 72.8 2.7 ± 0.6

(N = 4 follicles

Subordinate 2.0 0.4 7.4 ±+0.6* 19.1 ± 13.3* 13.2 ± 2.9*

(N = 8 follicles)

Day 11 (N = 4 follicles) 1.0 ± 0.0 b 16.0 ± 0.4c 9.2 4.7b 14.8 3.4 b
'bcComparison of mean values for Day 3, Day 7 (total), and Day 11; means within the same column with different superscripts differ significantly (p <
0.05).
*Different from dominant follicles on Day 7 (p < 0.01).

endocrinological conditions until follicles are almost fully
grown. In fact, in some species follicular development can
proceed to that point in the absence of the pituitary, sug-
gesting that follicular stages prior to the stage of recruit-
ment are regulated by factors internal to the ovary, rather
than by negative feedback interactions with the hypothal-
amus-pituitary [1]. However, follicles do not seem to have
the ability to develop through the final granulosa cell gen-
eration unless they are exposed to specific signals at the
time when they would normally become atretic. Consid-
eration of data from these three species suggests that the
number of granulosa cell generations that can be achieved
without special signals or hormonal support is positively
correlated with the size of ovulatory follicles in the species.
Antrum formation occurs at about the same follicular size
in the three species (around 0.2-0.4-mm follicular diame-
ter), but rats ovulate follicles that are 0.9-1.0 mm in di-
ameter, whereas preovulatory bovine and human follicles
are about 15 and 20 mm, respectively [1-3, 6]. Therefore,
in rats, recruitment of follicles into the ovulatory cohort
and most atresia occur around the time of antrum forma-
tion, whereas in cattle and humans, the stages with heaviest
atresia and the stage at which follicles are recruited into an
ovulatory cohort occur much later than antrum formation.
However, what is consistent among these species is that the
incidence of atresia is heaviest just before the final growth
to ovulatory size. In other words, in each species, follicles
seem to "hit a brick wall" that only a few follicles can get
over, but the size at which they encounter the brick wall
is positively correlated with the size of ovulatory follicles
for that species.
Why do most follicles become atretic when they reach
a certain species-specific size? Hirshfield [1] has suggested
that rat follicles reach the end of their normal life span at
the early antral stage (0.4-0.5 mm) because the metabolic
load on the avascular granulosa cell layer becomes too great
when the follicle attains that size. However, in species with
larger ovulatory follicles than those of rats, follicles can clearly
grow several granulosa cell generations beyond the point
when rat follicles normally become atretic. Although in these
species granulosa cell numbers increase with follicular di-
ameter, much of the additional increase.in diameter of the
follicle is due to expansion of the antrum. The fact that the
number of granulosa cell layers does not continue to in-
crease beyond a certain point in these species argues for
Hirshfield's hypothesis that access to oxygen and nutrients
is critically important for the granulosa cells. However, it
appears that follicles can solve the problem by enlarging
the surface area of the basement membrane while main-
taining a constant number of granulosa cell layers, thus
maintaining a particular ratio of basement membrane to
granulosa cell layers. Thus, the question of why follicles do
not continue to grow past a certain, species-specific size
unless they receive additional signals seems unanswered at
the present time.
The concepts and hypotheses presented in this section
are intended to lay groundwork for the discussion of how
some follicles escape atresia that follows. For more details
and additional references on the initial stages of follicular
development, the reader is referred to excellent recent re-
views by Hirshfield [1] and by Greenwald and Terranova
[7].
FOLLICULAR RECRUITMENT AND SELECTION
What are the mechanisms that allow only a small per-
centage of follicles to "climb the brick wall" (i.e., escape
atresia) and proceed to the final stages of follicular devel-
opment? Analysis of patterns of development of large fol-
licles in mammalian species shows that large follicles do
not develop at random, but their development occurs only
during particular reproductive states and/or during partic-
ular times of the reproductive cycle. During the 4-5-day
estrous cycle in rats, a cohort of follicles grows from a di-
228 FORTUNE
ameter of around 0.4-0.5 mm on metestrus to 0.8-1.0 mm
on proestrus [1]. In contrast, the growth of follicles to ovu-
latory size does not occur during pseudopregnancy or
pregnancy until the final 2-3 days, preceding the next es-
trus [1]. Likewise, in humans and other primates with men-
strual cycles, the development of large ovulatory-size fol-
licles is not random, but occurs at particular times of the
cycle [8]. Surprisingly little is known about the patterns of
follicular development in women with normal menstrual
cycles. Using ultrasonography to visualize large antral fol-
licles on both ovaries, Pache et al. [9] have determined
numbers and sizes of follicles throughout seven normal
menstrual cycles. Their report confirms what was com-
monly believed previously-ovulatory-size follicles do not
develop during the human luteal phase, but a cohort of
growing follicles emerges during the early follicular phase.
Only one follicle continues to grow during the late follic-
ular phase, and others in the cohort regress. Likewise in
pigs it appears that ovulatory-size follicles do not develop
during the luteal phase, although less direct information is
available for that species [7].
In contrast, other species exhibit patterns of follicular
development that involve the development of ovulatory-size
follicles throughout the cycle. This type of pattern has been
described most thoroughly for cattle. Large follicles do not
appear at random during the bovine estrous cycle. Ultra-
sonographic studies in which all follicles > 4-5 mm in an-
tral diameter have been followed over time have revealed
that either two or three times during the cycle a group of
3-6 follicles, termed a "wave," contemporaneously begins
to grow larger than 5 mm [10-12]. After several days, one
follicle is usually slightly larger than the others, and it con-
tinues to grow while the other, subordinate follicles re-
gress. Follicular waves begin around Days 2, 9, and 16 of
the estrous cycle in heifers with three waves of follicular
development and around Days 2 and 11 in heifers with two
waves [10]. The follicle that is dominant at the time of luteal
regression becomes the ovulatory follicle. There does not
seem to be any fundamental difference between cycles with
three vs. two waves of follicular development. The duration
of the luteal phase appears to determine, at least in part,
the number of follicular waves during a cycle. Estrous cycles
with three follicular waves have slightly, but significantly,
longer luteal phases than cycles with two waves [12,13],
and experimentally prolonging the luteal phase by treating
heifers with exogenous progesterone produced cycles with
4 or 5 waves of follicular development [14]. Follicular waves
also occur during pregnancy [15, 16] and during the pre-
pubertal period [17] (in contrast to the absence of ovula-
tory-size follicles during pregnancy, pseudopregnancy, and
the prepubertal period in rodents, as mentioned above).
Likewise, large follicles, similar in size to ovulatory follicles,
develop in horses during the luteal phase [18-20]. Most
mares have only one wave of follicular development per
cycle, which begins during the mid-luteal phase, but around
one-third of mares exhibit an alternative pattern of two waves
of follicular development, one beginning shortly after ovu-
lation and the other during mid-late luteal phase [20]. The
smaller size of ovulatory follicles has made it difficult to
discern patterns of follicular development in sheep by ul-
trasonography, and the regular patterns observed in cattle
have not been reported thus far, but it seems clear that in
sheep ovulatory-size follicles develop during the luteal phase
[21, 22].
Hence, there appear to be two different patterns of de-
velopment of large antral follicles in mammals (Fig. 2). In
one pattern, exemplified by rats, humans, and pigs, the de-
velopment of ovulatory-size follicles is suppressed, except
during the follicular phase of the cycle (and the last few
days of pregnancy or pseudopregnancy in species that have
postpartum estrus). In the other pattern, typified by cattle,
sheep, and horses, development of follicles to ovulatory or
near-ovulatory diameter is not confined to the follicular
phase, but occurs throughout the cycle. Dividing mamma-
lian species into these two different groups seems a useful
way to organize our current knowledge of follicular pat-
terns in mammals, but it may well prove too simplistic in
the future, as we learn more about follicular dynamics in
various species. Mechanisms that may subserve these two
different patterns of follicular dynamics will be discussed
below.
REGULATION OF FOLLICULAR RECRUITMENT
The term recruitment has been given to the growth of
follicles beyond the stage at which most follicles undergo
atresia. During recruitment, follicles try to scale the "brick
wall" that stands between them and ovulation. Recruitment
is not a random or isolated phenomenon; on the contrary,
follicles seem to be recruited as groups or cohorts, sug-
gesting that they have received a signal that allows them to
continue growth and development rather than regress. The
signal that stimulates recruitment appears to be a slight el-
evation in plasma FSH. There are various types of evidence
for that hypothesis. First, follicular recruitment is tempo-
rally correlated with slight increases in circulating FSH. Rats
exhibit a secondary surge of FSH on the day of estrus [23],
just before the next cohort of ovulatory follicles is re-
cruited. In primates, basal FSH is slightly higher at the be-
ginning of the follicular phase than during the luteal or late
follicular phases [24, 25]. In cattle, not only does a second-
ary surge of FSH on the day of ovulation precede the first
follicular wave of the cycle [26, 27], but also slight eleva-
tions in FSH have been shown to precede the second and
third follicular waves of the cycle [28] and the waves that
occur in prepubertal animals [17]. Relationships between
the dynamics of growth of large follicles and changes in
levels of basal FSH are less well documented in other spe-
cies and would be of interest.
Not only are there temporal correlations between ele-
vations in plasma FSH and the recruitment of follicles, but
 FOLLICULAR DEVELOPMENT IN MAMMALS
LH Surge Ovulation LH Surge Ovulation
r
u,
+1
c the typical number of ovulatory follicles for a given species.
0
0
0 their sister, subordinate follicles and prevent further follic-
l-
E known. If we hypothesize that follicles are recruited be-
a:
Granulosa Theca
FIG. 3. Patterns of change in levels of mRNA for steroidogenic en-
zymes and for oxytocin in theca and granulosa cells obtained from bovine
preovulatory follicles before the LH surge or between the LH surge and
ovulation. The two bars on the left side of each panel show levels of mRNA
in cells obtained during the early and mid-follicular phases, respectively,
and the bars on the right depict levels measured between the LH surge
and ovulation. (Data have been summarized from references 48-50). arom,
aromatase; HSD, hydroxysteroid dehydrogenase; scc, side chain cleavage.
also perturbations of those increases in FSH lead to con-
comitant changes in the patterns and/or number of re-
cruited follicles. Abolishing the secondary surge of FSH by
injections of follicular fluid containing inhibin prevents the
recruitment of the ovulatory cohort of the next cycle in rats
[29] and delays the first follicular wave of the cycle in cattle
[30]. In an experiment with cynomolgus monkeys in which
plasma LH was held constant and plasma FSH was in-
creased gradually, a pattern designed to mimic the early
follicular phase, increases in plasma estradiol gave evi-
dence that the small increase in FSH allowed the devel-
opment of follicles beyond the normal stage of atresia [31].
The more dramatic elevations of plasma FSH achieved dur-
ing hormonal regimes for superovulating women and do-
mestic animals clearly increase the numbers of follicles re-
cruited.
Since the emergence of waves or cohorts of follicles seems
closely tied to slight elevations in plasma FSH, one could
hypothesize that species in which ovulatory-size follicles do
not develop during the luteal phase (Fig. 2, lower panel)
have higher luteal-phase levels of feedback regulators like
estradiol and inhibin. Alternatively, hypothalamic/pituitary
229
sensitivity to such feedback may be greater in species in
which the development of dominant follicles is suppressed
during the luteal phase.
FOLLICULAR DOMINANCE
The number of follicles recruited is usually greater than
However, only a species-specific number of ovulatory fol-
licles continues to grow for more than a few days and reaches
ovulatory size. These follicles are called "dominant" folli-
cles because it is believed that once they are selected, they
in some way prevent further growth and differentiation of
ular recruitment. What determines which follicle(s) is se-
lected for dominance? The answer to this question is not
cause they are exposed to a slight increase in circulating
FSH just around the time when they would normally begin
atresia (i.e., they are in the right place at the right time),
then perhaps the follicle that is selected for dominance is
simply in exactly the right place (stage of development) at
exactly the right time and is better able to respond to the
slight elevation of FSH to continue its growth. But why then
don't the subordinate follicles also continue to grow; how
are they selected against?
Two hypotheses have been advanced to explain how the
dominant follicle exerts dominance. One hypothesis states
that the dominant follicle secretes something that directly
impairs further growth and development of subordinates.
In monotocous species, such a factor would clearly have to
be endocrine in nature, since it would induce regression
of subordinate follicles on both ovaries. Although this is a
reasonable hypothesis, and for a time the follicle regulatory
protein (FRP) described by DiZerega's group [32] seemed
a promising candidate for such a factor, subsequent re-
search did not support a role for FRP in follicular domi-
nance (personal communication from Sharon Tonetta). Al-
ternatively, the dominant follicle could cause the regression
of subordinates indirectly, via negative feedback mecha-
nisms. According to this hypothesis, the secretion of feed-
back regulators, such as estradiol and inhibin, by the dom-
inant follicle (or perhaps by the whole cohort of follicles
during the first few days after recruitment) would cause a
decrease in FSH to levels that would not support the fur-
ther growth of subordinates. How would the dominant fol-
licle escape inducing its own atresia? The dominant follicle
may have reached a stage of differentiation in which it can
sustain growth in the presence of lower levels of circulating
FSH. Experiments in Zeleznik's laboratory support this idea.
Using the model mentioned above, in which both FSH and
LH are experimentally regulated in cynomolgus monkeys,
Zeleznik and Kubik [31] showed that levels of FSH that are
insufficient to recruit follicles are sufficient to maintain fol-
licular growth once it has been initiated. The ability of the
 230 FORTUNE
dominant follicle to continue growth and development in
the face of lower circulating levels of FSH may be due to
increased blood flow through the follicle and/or to the ac-
quisition by the dominant follicle of LH receptors on the
granulosa cells [33]. It does appear that FSH remains crit-
ically important to the dominant follicle, even during the
dominance phase, since experimental reduction of plasma
FSH during that time in cattle is correlated with cessation
of growth, and in some animals the demise, of the domi-
nant follicle [34].
Why do subordinate follicles exist if they simply are ob-
literated by the dominant follicle? Clearly there is much
wastage during follicular development. However, having a
steady progression of follicles reaching the stage at which
a slight elevation in FSH could recruit them for further
growth ensures that when the slight elevation in FSH oc-
curs, follicles will be recruited promptly. And the recruit-
ment of more follicles than the ovulatory number for a spe-
cies ensures that at least one will be at exactly the right
stage of development to capitalize quickly on that hor-
monal spur to its further development. Experiments by Ko
et al. [35] have shown that if the dominant follicle of the
first follicular wave during the bovine estrous cycle is de-
stroyed during the first few days of the wave, regression of
the largest subordinate follicle is significantly delayed, but
destruction of the dominant follicle later during the wave
is followed by early recruitment of the next wave. There-
fore, the recruitment of "excess" follicles can ensure that
if something happens to the lead follicle early in its dom-
inance phase, another follicle can quickly fill the gap. Pre-
sumably destruction of a dominant follicle leads to a slight
increase in FSH that allows the leading subordinate to be-
come dominant, but such an increase in FSH has not (to
my knowledge) been shown experimentally.
DIFFERENTIATION OF DOMINANT FOLLICLES
The follicle(s) selected for dominance from each cohort
or wave not only continues to grow, but also differentiates
functionally in ways that prepare it for ovulation and also
prepare the female for potential pregnancy. The secretion
of increased quantities of estradiol by the selected folli-
cle(s) appears to be of primary importance and sets it apart
from its sister subordinate follicles. In most mammalian
species that have been examined, follicular estradiol syn-
thesis requires the cooperation of both follicular endocrine
cell types and both gonadotropins, with theca cells pro-
ducing androgens in response to LH and granulosa cells
aromatizing androgens to estradiol in response to FSH, and
later in follicular development in response to both FSH and
LH [36]. Increased capacity to secrete estradiol appears to
arise from increases in the ability of theca cells to respond
to LH by secreting androgen and of granulosa cells to aro-
matize androgen to estradiol. In this section, I will concen-
trate on the most basic developmental changes in steroido-
genic function as dominant follicles differentiate toward
ovulation, and on the roles of the two pituitary gonadotro-
pins in these changes. However, there is much evidence
that other factors (e.g. growth factors, steroids, inhibin, and
inhibin-related proteins,etc.) also play important modula-
tory roles in follicular function, and the reader is referred
to recent reviews for information on these factors [37, 38].
In this discussion of the steroidogenic differentiation of
dominant follicles as they develop towards ovulation, I will
use rodents (primarily rats) and ruminants (primarily cat-
tle) as examples of the two types of patterns of follicular
recruitment depicted in Figure 2. The differentiation of rat
follicles during the follicular phase has been reviewed sev-
eral times in the last few years. The reader is referred to
those reviews for additional details [36, 39]. In brief, small
but sustained elevations in circulating LH are crucial for the
development of ovulatory follicles. LH stimulates further
differentiation of the theca cell layer, including an in-
creased capacity to secrete androgen. Aromatization of an-
drogen by granulosa cells increases the production of es-
tradiol, which is crucial for the further differentiation of the
granulosa cells.
Therefore, it appears that in rats, once the ovulatory co-
hort of follicles has been selected, the differentiation of the
follicle for increased estradiol secretion proceeds rapidly
over the course of 2-3 days. However, in cattle, which de-
velop dominant nonovulatory follicles as well as dominant
ovulatory follicles, the differentiation of ovulatory follicles
seems to occur in two stages. Dominant, nonovulatory fol-
licles obtained during the course of the first wave of fol-
licular development in cattle exhibit increased estradiol and
decreased androgen in follicular fluid compared to sub-
ordinate follicles obtained from the same ovaries (Table 2:
Day 7 [40, 41]). Theca cells from dominant follicles secrete
significantly more androgen and granulosa cells have greater
capacity to convert androgens to estradiol than follicular
cells from subordinate follicles [40]. Therefore, even dom-
inant follicles that are selected during the luteal phase de-
velop an enhanced capacity for thecal androgen and gran-
ulosa estradiol production. Presumably androgen is low in
the follicular fluid of healthy dominant follicles because an-
drogen secreted by the theca is used as a precursor for
estradiol synthesis by granulosa cells. If progesterone re-
mains at luteal levels, these dominant follicles do not con-
tinue to increase estradiol secretion (Table 2: Day 11 [40, 41]).
Estradiol secretion decreases, as indicated by decreases both
in the plasma and follicular fluid, and also in androgen and
estradiol production in vitro. This decrease in negative
feedback from the ovary, as a dominant, nonovulatory fol-
licle begins to regress, presumably allows the next small
increase in basal FSH, which occurs too late to rescue the
faltering dominant follicle, but induces the next round of
recruitment.
If, on the other hand, luteal regression occurs during the
recruitment or dominance phase, the follicle is exposed to
 FOLLICULAR DEVELOPMENT IN MAMMALS
additional hormonal signals that allow it to develop fully,
to the point where it is secreting enough estradiol to elicit
the LH/FSH surge and ovulation. When granulosa and theca
cells were obtained at 0, 12, 24, or 48 h after heifers re-
ceived injections of prostaglandin F2a to induce luteolysis
and initiate a follicular phase, the ability of theca cells to
respond to LH with increased androstenedione production
and the capacity of granulosa cells to aromatize androgen
to estradiol increased dramatically, especially in the interval
between 12 and 24 h after the initiation of luteolysis [42, 43].
In cattle, the decline in plasma progesterone at luteal
regression is followed by small increases in basal LH, and
increases in LH pulse frequency [27]. The enhanced thecal
responsiveness to LH and enhanced aromatization capacity
of the granulosa cells are presumably a result of this small
elevation in circulating LH.
The importance of LH in the final stages of differentia-
tion of the follicle prior to the LH surge is further empha-
sized by the results of experiments in which plasma pro-
gesterone was artificially maintained at levels that were
intermediate between luteal and basal for prolonged pe-
riods of time. Under these conditions, LH pulse frequency
increased to follicular phase levels [44, 45], growth of the
dominant follicle continued in a linear fashion, and plasma
estradiol was elevated for much longer than normal [14, 45].
Although the level of plasma progesterone achieved in these
experiments was low enough to permit the follicle to
undergo follicular phase-type differentiation, it was appar-
ently high enough to block induction of the LH surge. This
experimental situation underscores the power of LH to drive
follicular development, since it temporally extended and
functionally exaggerated the follicular phase. The results have
also suggested that the lower fertility associated with the
use of low-dose progestins to synchronize the estrous cycles
of cattle may result from the prolonged development of the
ovulatory follicle and perhaps from the prolonged expo-
sure of the oocyte and/or reproductive tract to elevated
levels of estradiol [45, 46].
Therefore, whether follicular waves are continuous or
occur only during the follicular phase, a salient feature of
the differentiation of dominant follicles is an increase in the
ability of the follicle to produce androgens and estradiol.
The differentiation of the dominant follicle into an ovula-
tory follicle capable of eliciting an LH surge appears to be
due to the ability of slightly increased plasma LH to further
increase the ability of the follicle to produce androgen and
estradiol. Paradoxically, these changes in steroidogenic ca-
pacity that are induced by small increases in basal LH, in
the presence of basal concentrations of FSH, are reversed
by exposure of the follicle to surge concentrations of the
gonadotropins. The ability of theca cells to secrete andro-
gen decreases after the LH/FSH surge, as does the aro-
matizing capacity of granulosa cells [36]. At the same time,
progesterone production increases, and in cattle the ability
231
of granulosa cells to secrete oxytocin is greatly increased
by exposure to the LH surge [47].
Changes in steroidogenic capacity of differentiating rat
ovulatory follicles appear to be mediated by correlative
changes in levels of messenger RNA (mRNA) for appropri-
ate steroidogenic enzymes. Studies with rat follicles have
shown that levels of mRNA for 17ot-hydroxylase and aro-
matase increase during the development of antral follicles
before the LH surge [36, 39]. Levels of these messages de-
crease precipitously in follicles that have been exposed to
surge levels of LH, obtained between the gonadotropin surge
and ovulation. In contrast, levels of mRNA for P450 side
chain cleavage increase in response to surge levels of go-
nadotropins, and this message becomes constitutively ex-
pressed, in concert with the shift in production of proges-
terone during the luteal phase. Likewise in cattle, thecal
mRNA for 17oa-hydroxylase and granulosa cell mRNA for
aromatase also decrease precipitously after the gonadotro-
pin surge, concomitant with an expected increase in oxy-
tocin mRNA [48, 49] (Fig. 3). However, bovine follicles ob-
tained after the LH/FSH surge exhibited an unexpected and
puzzling decrease in mRNA for enzymes involved in pro-
gesterone biosynthesis [50] (Fig. 3). These decreases stand
in contrast to the increased levels of progesterone in fol-
licular fluid and increased ability of theca and granulosa
cells obtained between the gonadotropin surge and ovu-
lation to secrete progesterone in vitro [42, 51].
It is clear that further exploration of the endocrinolog-
ical differentiation of follicles in response to the LH/FSH
surge would be useful. The mechanisms that enable basal
and surge concentrations of LH and FSH to produce com-
pletely different results are not understood and also pres-
ent fruitful ground for further studies.
ACKNOWLEDGMENTS
I am grateful to Drs. AN. Hirshfield, JJ. Eppig, and J. Sirois for reading this
manuscript and for their helpful comments, and to Dr. Hirshfield for providing Fig-
ure 1.
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